Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the near selective loss of neurons in the substantia nigra. While the etiology of the disease is unknown, genetic mutations responsible for familial forms of PD will likely provide critical molecular clues to the underlying mechanisms of idiopathic disease. Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. Parkin is now widely recognized as a pro-survival protein rapidly up-regulated during cell injury. Parkin is believed to possess broad ranging effects on mitochondrial biology that are likely relevant to idiopathic PD. However, the biochemical substrates of these effects are largely unknown. The long-term goal of our work is to uncover the molecular machinery responsible for parkin's influence on mitochondria. This information will not only improve our understanding of an important neuronal stress response protein but will also elucidate new biochemical pathways likely involved in the selective neurodegeneration in PD. The experimental focus of this application is on the mitochondrial defects that result from neuronal deficiencies in parkin and identifying the proteins responsible. There are three specific aims. 1) Analyze the parkin-dependent ubiquitination of two novel parkin substrates involved in mitochondrial fission and apoptosis. We will determine the effects of endogenous parkin on protein half-life and localization, and the contribution of these candidate substrates on mitochondrial defects in parkin deficient neurons. 2) Analyze the mitochondrial consequences of parkin deficiency in primary cultured neurons, and examine enhanced vulnerabilities of parkin null neurons to stressors implicated in idiopathic PD. 3) Examine mitochondria-specific changes in protein trafficking and function that occur in parkin deficient neurons. The completion of these studies will establish mechanistic links between the E3 ligase activity of parkin and its function as a pro-survival stress response protein with mitochondrial influence. This work will elucidate novel biochemical pathways involved in the pathogenesis of PD and potentially uncover innovative targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Parkin is critically involved in a protective neuronal stress response and is causally linked to familial Parkinson's disease. The goal of this study is to understand the precise mechanisms by which parkin maintains integrity of the mitochondria and to define molecular events that can cause Parkinson's disease. These pathways, once discovered, may identify new therapeutic targets for the treatment of this devastating neurological disorder.